Effects Of Sex And Aging On The Immune And Epithelium Cells Of Bladder By Single-cell Transcriptomic Analysis

Background and objectiveThe epidemiological characteristics,clinical manifestations,treatment effects,and prognosis of bladder diseases have obvious sex dimorphism,especially in the elderly.It is unclear how and why these bladder disorders manifest differently in the sexes with age.These bladder diseases with age have a chronic inflammatory component and overlapping lower urinary tract symptoms(LUTs),and some have been found to be associated with basal cells.The bladder is not only a urine storage organ,but also an important mucosal barrier for the body.The bladder usually lacks specialized mucosal secondary lymphoid organs(SLO)during development,and forms a unique and relatively static mucosal immune system.In addition,another feature of the bladder mucosa is that under steady-state conditions,basal stem cells renew very slowly,but rapidly regenerate and differentiate when pathogens invade and epithelial integrity is disrupted,together with resident immune cells to promote inflammation and rapidly repair damages.However,studies on the interaction of immune cells with bladder epithelial cells or stromal cells are rare.With the development of sc RNA-Seq,it has become a sword for the study of aging.Therefore,we used Sc RNA-seq to depict a comprehensive immune and epithelial cell atlas of bladder by Sex and Aging,and to reveal how the immune system and epithelium integrates numerous interconnected components,pathways,and cell types in sex and aging.This study aims to find the potential mechanisms of sex dimorphism in bladder aging and aging-related diseases.MethodsAfter sacrificed,12 mice(C57BL/6J)was divided into old female team(OF),old male team(OM),young female team(YF),and young male team(per n=3).We made the bladder into the single-cell suspension,then first selected immune cells by flow cytometry.Then,the samples were mixed according to the ratio of Cd45+immune cells:total bladder cells≈1:5,and the library was prepared and sequenced.Then we used all kinds of R packages to analysis DEGs,DEG intersection,gene function annotation Gene Ontology and KEGG signaling pathway analysis,pseudo-temporal analysis and cell-cell communication analysis.Results83574 bladder cells from 12 mouse were divided into 12 clusters,including T lymphocytes(TC),B lymphocytes(BC),myeloid lymphocytes(Meyloid),plasma cells,epithelial cells,mesenchymal cells(fibroblasts,myofibroblasts),smooth muscle Cells,SMC),Vascular Endothelial Cells(BEC),Lymphatic Endothelial Cells(LEC),Adipocytes,Schwann Cells.Then the clusters were divided into all kinds of sub-groups.It was also found that the senescence-related bladder cells were GZMK~+CD8~+T cells,senescence-associated B lymphocytes(aged-associated B cells,ABCs),and Il17a~+ILC3s.Aging increased the proportion of Gzmk~+Cd8~+T cells,Zbtb32~+ABCs,Il17a+ILC3s,but there were no sex difference.Bladder na?ve B cells in young female mice bladder were higher than other groups.Aging increases the activation and exhaustion signals of T and B lymphocytes in aged mice with sex difference.Cd4~+and Cd8~+T cells in old female were significantly enriched for Gzmk.Aging increases the activation of B lymphocytes,antibody-secreting of plasma cell and IL-6 secretion in the bladder of old female.Aging increased ILC3 in old female bladder with the differentiation of Th17 cells,C-type lectin receptor signaling and the signaling pathway of cell senescence,while the main role in old male was NK,with TNF mediated signaling pathway and inflammatory signaling pathways.Aging increased the processing and presentation of MHC class I antigen peptides and exogenous antigens of DC in female,while it increased cytokine production,complement activation and signaling pathways of the P38 MAPK cascade of DC in male.Aging increased inflammatory signalings such as TNF,cytokines,ROS and NO mediated signaling pathways in male.Aging increased the transcriptome differences in circulating basal cells and EMT,basal cells.Basal cells have three cell subtypes,BC1,BC2,and BC3,and have different regeneration and differentiation potentials.Aging increased the apoptosis signaling pathway mediated by differentially expressed genes(DEGs)and the immune response pathway mediated by cytokines in bladder basal cells with sex-difference,while down-regulation differentially Genes mediate the cell growth,differentiation and proliferation signaling pathways.Although the signaling pathways of bladder basal cells affected by aging are similar between male and female,the mechanism was sex-dimorphic.Male and female share the same senescence expression pattern Mif-Cd74＿Cd44 and Mif-Cd74＿Cxcr4 receptor-ligand pairs in B cells.Aging results in an interaction that is present in male but not female bladders,with Cd40lg-Itgam＿Itgb2 of ILC-DC and ILC-MC in old male but absent in old female bladders.In general,older male mouse bladders have more ligand-receptor pairs and signaling pathways than female.KIT signaling in Basal-B cells is one of the few that is enhanced in female mouse bladders,and this signaling pathway may provide new insights for the diagnosis and treatment of female autoimmune diseases and IC/BPS.Aging increased the cell-cell communicaton in old males,including MIF,CCL,CXCL,TNF and GALECTIN signaling pathways of MC,DC,and Cd4~+T cells.ConclusionsIn this study,we profiled the the transcriptomic atlas of immune and epithelium cells of bladder sampled from male and female of two distinct age groups(YM,YF,OF,OM).Then,we investigated sex-and age-related differences in the immune and epithelial cell composition of bladder,molecular programs,and cell-cell communication network.Obvious differences in all of these aspects were observed between sexes,particularly with aging,which also provides potential mechanisms for sex-based differences in aging-associated bladder diseases.Overall,this work expands our knowledge of sex differences in immune and epithelial cells of bladder.